Stem Cell Implants: Emerging Innovation for Stroke Recovery

Stroke is a debilitating neurovascular injury that those effects hundreds of thousands of Americans each year. Despite the high prevalence, disease morbidity and mortality, options for stroke intervention and rehabilitation are still limited. Stem cells have shown promise in stroke treatment due to their ability to self-renew and differentiate into different cell types. The primary sources of stem cells used today are bone marrow and fetal brain tissue, with mesenchymal stem cells, bone marrow stem cells and neural stem cells being particularly well-studied. By secreting therapeutic and neurogenic substances they are hypothesized to help foster recovery at the site of injury. Delivery mechanisms for stem cell therapy include intracerebral, intra-arterial, intraperitoneal, intravenous, intraventricular and intranasal routes with radiographic imaging now being used to monitor the progress of stem cell therapies. Stem cell implants have been found to be safe but optimal treatment strategies are still being established with several promising studies underway. Future efforts should continue to focus on improving efficacy, exploring alternative stem cell sources, enhancing migration capability and survival and educating stroke patients on the benefits and risks of stem cell therapy.


Introduction
More than 795,000 strokes occur in the United States every year, with nearly 1 in 4 happening in those with a prior history of stroke [1]. It is estimated that the strokerelated healthcare costs were approximately $53 billion between 2017 and 2018 [1]. Furthermore, stroke is a devastating disease in which survivors frequently experience motor Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CCBY) license (https://creativecommons.org/licenses/by/4.0/). and neurological deficits along with a decreased quality of life [2,3]. Due to its high prevalence and disease morbidity, the treatment and management of stroke remains an area of great therapeutic consequence.
The many varying etiologies of stroke correspond to different treatments which depend largely on the type and timing of the infarctive event. There are two different subtypes of stroke: ischemic and hemorrhagic. Ischemic strokes result from an occlusion of the blood supply whereas hemorrhagic stroke is defined by bleeding from a vessel [4]. Ischemic strokes account for approximately 87% of all strokes and uniquely can be treated with an intravenous injection of tissue Plasminogen Activator (tPA) [1,5]. This promotes the dissolution of clots and restores patency to the occluded vessel. However, this intervention has a limited time window and must be administered within 3-4.5 hours of insult [5,6]. Additional therapeutic options include antiplatelet therapy, which has been shown to be effective at preventing a stroke recurrence within 48 hours but cannot ameliorate the ischemia that has already occurred [6]. Surgery and other minimally invasive interventions are also used in the setting of ischemic stroke and include procedures such as mechanical thrombectomy, ventriculostomy and decompressive craniectomy [7,8]. Mechanical thrombectomy is not indicated in all patients and additionally suffers from time constraints as well as it is only effective up to 24 hours after initial presentation [9]. Unfortunately, there are few treatment options available to address hemorrhagic strokes. These involve the management of hypertension and raised intracranial pressure, hemostatic therapy and surgical intervention [10,11]. All of these treatments are largely focused on damage control and preventing spread or worsening of damage from the inciting event. However, once Central Nervous System (CNS) neurons are damaged, they are for the most part, unable to regenerate, with no current treatments available intended to renew the damaged neural networks [12]. New research is hoping to bridge this gap in therapy, with stem cells offering a promise in neuroprotection and neuroregeneration in the management of stroke.
Research into stem cells is still very much in its infancy. Less than 30 years ago the first primate embryonic stem cell line was derived from rhesus monkeys and it was only in 1998 that the first human embryonic stem cells were derived by Thomson, et al., [13,14]. Since then, the field has exploded with a variety of applications scoping many different domains of medicine [15]. There are three classes of stem cells that are being explored in the treatment of stroke. These include neural stem cells, bone marrow stem cells and mesenchymal stem cells [16].

Mechanism of Action
Stem cells are cells that uniquely have the ability to self-renew and create functional tissues. More specifically, they can differentiate into nearly any cell type present in the human body, which is what drew scientific interest to their therapeutic potential in the first place. There are several major categories of stem cells: (1) Totipotent, (2) Pluripotent and (3) Multipotent [17]. Totipotent cells can form all cell types within the human body, plus the extraembryonic cells that are crucial for early development of the fertilized embryo. Pluripotent stem cells are commonly used in therapies as they can give rise to all cell types within the human body.
Multipotent stem cells are similar, but more limited in their differentiation potential-these cells include the adult stem cell and the neural stem cell. Stem cells are further distinct from precursor cells, which are limited to differentiating into a single cell type and progenitor cells, which cannot divide indefinitely [18,19].
Several mechanisms for the action of stem cells have been proposed over the years. Initially, it was thought that grafted cells could replace dead sections of the brain and become integrated in their place. This hypothesis has given way to the modern theory of the bystander effect, wherein implanted cells secrete therapeutic and neurogenic substances that ameliorate injury and foster regeneration [20,21]. Ever since ischemic stroke was also recognized to impact non-neuronal cell components of the brain such as glia and vasculature, stem cells are also thought to contribute to angiogenesis, vasculogenesis, antiapoptosis and anti-inflammation [22].
Primarily due to its well-established safety in the literature, the bone marrow is the primary source of stem cells in use today [23,24]. Bone marrow derived populations include Mesenchymal Stem Cells (MSCs), bone marrow stem cells (BMSCs), Mononuclear Cells (MNCs), endothelial progenitor cells (SB623), Multipotent Adult Progenitor Cells (MAPCs) and Multilineage-differentiating Stress-Enduring cells (MUSE) [18]. Another primary source of stem cells is human brain fetal tissue, which has been used to derive important cell lines of Neural Stem Cells (NSCs) [25].
Several initial studies determined the various neuroprotective and neuroregenerative effects of these cells in preclinical and later clinical settings [26][27][28]. Based on these trials, MSCs, BMSCs and NSCs are particularly well-studied and promising. For example, as an autologous cell with determined population purity and well-defined viability and multilineage differentiating potential, MSCs have been shown to promote neurogenesis and anti-inflammation, possibly mediated through the secretion of factors including IL-1, TNF-alpha, IL-11 and TGF-β [20,29,30]. Studies on murine stroke models have also demonstrated the ability of MSCs to decrease levels of axon growth inhibitors, leading to an increased density of functional axons in the ischemic area [31,32]. BMSCs exert similar mechanisms of action as secreted factors play roles in neurogenesis rather than neuronal replacement. Unfortunately, neither MSCs or BMSCs studies have led to significant and replicable improvements in neurological outcomes of stroke patients, but efforts continue. NSCs working through neuroregenerative effects too have shown promise in preclinical models and there is some data to suggest that implantation of the cell line CTX0E03 has resulted in valid improvements in functional recovery [33]. Notably, to some extent, all three cell lines have shown the ability to migrate to damaged areas and even to guide the migration of endogenous cell via "biobridges" of matrix metalloproteinases in-vivo models [34,35]. However, these clinical studies are very much in early stages of research; efficacious and generalizable criteria for stem cell administration are still lacking.
The future of stem cell therapies is multifaceted. Naturally, clinical trials with different lineages of cells are underway, but beyond this classic approach, it has been proposed recently to use derivatives of stem cells such as vesicles, mitochondria, exosomes and micro-RNAs to foster regeneration in the ischemic area (Fig. 1). These cell components have been shown to exert similar paracrine effects as compared to stem cells, resulting in neurogenesis and angiogenesis [36,37]. In a similar vein, it has been proposed that if stem cell therapies are considered as biologics with regards to their paracrine effects, it may be easier to optimize delivery routes, classify the stem-cell secreted factors and eventually design drugs that mimic these effects [38].

Delivery Mechanisms
Unsurprisingly, several different approaches for stem cell delivery exist in the laboratory setting and many have been translated to clinical settings. Different administration routes have included intracerebral, intra-arterial, intraperitoneal, intravenous, intraventricular and intranasal [39]. Stem cells delivered intravenously and intrathecally in stroke patients has led to improvements in terms of aphasia, motor deficits and spasticity [40]. The advantage of more invasive routes such as intracerebral is that they allow for a larger number of implanted cells to reach target destinations, but suffer from limited generalizability, immune rejection and increased risk for infection. On the other hand, less invasive routes such as intravenous do not suffer from these disadvantages to the same extent and have even shown to have similar effectiveness compared to other delivery methods to the point that most modern clinical trials prefer to utilize this technique [41,42]. However, one of the major drawbacks of intravenous stem cell therapy is the pulmonary first-pass effect, meaning the total amount of stem cells injected does not reach the site of injury. Depending on the type of stem cell, very few reach the target, thus diminishing the therapeutic effect [43]. There have been recent strides in developing more targeted stem cell delivery methods, not only in terms of reaching the location of injury but also addressing specific cellular defects following a stroke (Fig. 2). Extracellular Vesicles (EVs) or exosomes are extracellular membrane-bound vesicles that transmit cargo without direct cellular contact as a form of intercellular communication [44][45][46]. EVs have regenerative properties when derived from mesenchymal stem cells [47]. Different exosome treatments that involve altering genetic properties have improved recovery of the damaged tissue. For example, miR-542-3p is decreased following stroke in mice models and is thought to play a role in inflammation along with TLR-4 [48]. An exosome treatment in which miR-542-3p was overexpressed and injected into a mouse stroke model paracele led to decreased brain injury as well as decreased inflammation [48]. In a similar vein, neural stem cell exosomes treated with interferon gamma exhibited stronger therapeutic effects in rats when stereotactically transplanted into the area of infarct when compared to the group receiving the untreated neural stem cell exosomes. Interferon gamma was used to condition the exosomes due to its ability to improve cell survival in conditions of oxidative stress [49]. Next, rat stroke models administered miR-17-92 cluster-enriched exosomes from mesenchymal stem cells intravenously had increased neurogenesis and neuroplasticity along with oligodendrogenesis [50]. Further investigation regarding various ways to induce multiple genetic modifications to precondition exosomes may enhance neurorestorative effects.
Much of the current stroke therapy literature turns to EVs as personalized targeted delivery vehicles especially in the application of stroke where multipotent mesenchymal stromal cells have shown therapeutic effects [51,52]. An important aspect of NSC therapy is the regulation of NSCs to differentiate into the desired cell fate after transplantation on neural tissue [53][54][55][56]. It is known that many of the stem cells transplanted to the recipient host will commit to the glial progenitor lineage which limits the therapeutic effects of NSCs in the setting of post-stroke and reperfusion therapy. Although delivery via exosomes may serve to improve some of the effects of stroke, they are not able to target the site of injury and are therefore limited in terms of their benefits. Nanoparticle delivery systems are one strategy developed to overcome this. One study involved using iron oxide nanoparticles-harboring mesenchymal stem cells to enhance delivery to the ischemic lesion in rats. Following systemic injection into the tail vein, the rats were fitted with a 3D-printed helmet containing a magnet which localized the nanoparticles to the area of the lesion. This group had enhanced blood vessel density and decreased damage to the neurons in the location of the lesion [57]. Lin, et al., demonstrated that the utilization of theranostic nanomedicine leads to a 3-4-fold increase in neuronal differentiation of stem cells that can be detected in vivo with Magnetic Resonance Imaging (MRI) [53]. These findings suggest the important role of nanomedicine in clinical therapy. Additionally, there is evidence to suggest that mitochondria may serve as regulators of NSCs which can additionally play a role in targeted gene therapy [58].
Another obstacle has been the differentiation of neural stem cells into astrocytes rather than neurons. The use of superparamagnetic iron oxide nanoparticles and small interfering RNA/antisense oligonucleotides against Pnky lncRNA in combination with neural stem cells has shown promise in guiding differentiation into neurons as Pnky lncRNA inhibits neuronal differentiation [59]. When this system was employed in mice by injecting the stem cells directly into the area of infarct, the infarct volume was lower than that of mice given neural stem cells without this genetic alteration [53]. Another method for increased therapeutic effect includes the use of biodegradable polymeric nanoparticles containing adipose stem cells genetically modified to overexpress vascular endothelial growth factor [60].

Outcomes Thus Far
The safety of stem cell implants in stroke patients has been well established in several phase 1 and 2 clinical trials [29,30,33,. No severe adverse events attributable to stem cell therapy have been reported. Adverse events that have been attributed to treatment have been minor, transient and primarily due to route of delivery (e.g., headache from intracranial injection) [ A recent meta-analysis of randomized trials with scores on the mRS, NIHSS and BI at 6, 12 and 24 months found significant differences in NIHSS at 6 months and mRS at 12 months that favored treatment [83]. Although differences in the other measures were not statistically significant, the authors were only able to pool data from four studies (with each analysis containing no more than three), resulting in only between 50 and 81 patients being analyzed for each outcome. This small overall sample along with significant heterogeneity between studies (in part due to inclusion regardless of cell type, route and clinical setting) resulted in wide confidence intervals that, while unable to exclude the null, were still consistent with substantial functional improvement with treatment [83].
Given the large number of parameters that can vary in this space (cell type, dose, route of delivery, rounds of treatment, time since stroke onset, etc.), the number of possible best use cases is vast. Different methods may be better suited in the acute versus the chronic setting and stacking the optimal treatment in each setting may have significant synergistic effects. The established safety of the treatment, along with the exciting possibilities warrants further study and a number of ongoing trials are doing just that Table 1 shows trials currently ongoing in this space along with cell type, route of delivery, clinical setting, trial phase, sample size, trial design and estimated completion date.

Radiographic Findings
The promise of stem cell therapies has mandated the study of how cells are trafficked and utilized in the body. In the preclinical setting, much of this information has come from dissection and analysis of frozen specimens. Imaging as a noninvasive method of monitoring therapeutic efficacy has been explored more recently. Classically there are two different approaches to tracking stem cells: direct and indirect labeling with markers such as super paramagnetic iron oxides, perfluoropolyether and perfluorocarbon which can be picked up by MRI or Positron Emission Tomography (PET) scanning [84]. Several studies have investigated these techniques, but challenges have included the limitations of spatial resolution of current imaging modalities and the dynamics of labeling, such as the quantification of tracer uptake and the concentration of tagged cells in a sample [85][86][87][88]. One recent development aimed at managing these challenges is the introduction of reporter genes in stem cells, which when expressed form molecules that can bind to specific injected radiotracers allowing for easier visualization of stem cell migration [89].
Neuron-derived EVs regulate the Blood-Brain Barrier (BBB) in both physiological and pathological states [90]. Because EVs can cross the BBB, they may serve a role when looking for stroke biomarkers or radiological imaging. Some studies already utilize CD9 or CD63 as a specific marker for EVs that can be fluorescently tagged with GFP in neuroglia and tracked for radiographic studies [91,92]. Fig. 3 highlights the main functions of exosomes which are being utilized in research as drug therapy vehicles such as in the setting of stroke. In animal models, Neural Stem Cells (NSCs) in EVs were shown to improve functional outcomes post-thromboembolic stroke [93]. Exosomes have promising roles in clinical therapy while also allowing for the visualization of using biomarkers to understand their intercellular interactions and therapeutic effects [45,46].
Radiographic assessment is important for localizing neurological defects and understanding the sequelae of stroke. In acute settings of suspected stroke, Computed Tomography (CT) can be utilized due to fast imaging speed, wide availability and production of good contrast between a bright clot and surrounding lower attenuating cortical tissue and Cerebrospinal Fluid (CSF) [94][95][96]. MRI can also be utilized but takes longer to obtain imaging [94][95][96].
In one study, radiographic results administering EV NSCs in the context of stroke showed decreased infarct volume [93]. This decrease was shown in settings where the EV NSCs were administered both within and outside the tPA clinical therapeutic window [93]. To elaborate on this finding, other studies show coupling of angiogenesis and neurogenesis in settings of neurovascular ischemia [97,98]. It is known that when embryonic stem cells are introduced into rat brains during times of focal cerebral ischemia, these cells can be tracked in-vivo migrating along the corpus callosum to reach these ischemic areas from distances as far as the opposite hemisphere [99]. Porcine studies utilizing EV NSCs also show significant neuroprotection from induced ischemic stroke [100]. MRI results comparing the NSC treatment group from placebo show at the 84-day mark significant tissue level recovery, decreased cerebral lesion volume and preservation of white matter integrity [100]. The current research utilizing animal models shows evidence of potential clinical use of NSCs in the setting of acute ischemic stroke which can be monitored radiographically for spatial and temporal changes.
As discussed in the previous section, initial data regarding clinical Phase I/II trials of the utilization of mesenchymal stem cells in chronic stroke shows promise. In two of these studies, MRI results showed no progression or new ischemic lesions in the brain between the delivery of NSCs and prior to discharge [64,101]. Interestingly, one trial showed a reduction of mean lesion volume >20% at the one-week mark post mesenchymal stem cell infusion as evident on MRI [64]. These early clinical findings show much promise in the therapeutic utilization of NSCs in the treatment of ischemic stroke.

Areas Needed for Further Discovery
In the present study, we critically appraised and critiqued the wealth of emerging cell therapy research for stroke recovery. Though much has been achieved, several areas for focused inquiry and effort have been identified. The most pressing is to establish the efficacy of Stem Cell Therapy (SCT) as a therapeutic tool, as discussed earlier. However, additional areas for exploration include developing alternative stem cell sources and wider patient education on SCT.
Intracerebral transplantation of BMSC and MSC implants has demonstrated early promise in promoting the neurogenesis process post-stroke, but both remain steadily inefficacious relative to NSC implants [18,102]. Current evidence suggests that further exploration of BMSC and MSC, as well as Umbilical Cord Derived Mesenchymal Stem Cells (UC-MSC), represents a worthwhile pursuit in light of their immunomodulatory properties, which may prove clinically valuable for tackling prolonged post-stroke inflammation states [18,103]. The endogenous repair mechanisms promoted by MSCs have been specifically attributed to recruitment of immune mediators IL-6 and PGE2 plus responsiveness to IL-1β, which enhances monocyte and granulocyte recruitment for functional reduction of infarct volume [42,104]. However, despite evidence indicating the safe implantation of such stem cell populations in human replacement trials, establishing efficacy and best use cases with narrowing indications is the biggest challenge for future research [18,42,103]. There also exists a surge of early data proposing new expandable cell lines and methodologies with marked pre-clinical potential for neuropathologies [105][106][107]. These and the above-described findings taken together, future stroke research should concentrate efforts on enhancement of stem cell migration capability and survival within the ischemic environment.
Moreover, patient attitudes and understanding surrounding SCT also represent a muchdesired focus area. The available literature suggests that stroke patients are stratified in their attitudes towards SCT, which may conceivably limit their willingness to participate in clinical trials or, perhaps worse yet, motivate participation without consideration of the associated risks [108][109][110]. Akid and colleagues reported that in a controlled sample of 84 ischemic stroke patients, 12% (10/84) had prior knowledge of SCT but upwards of 36% (30) reported a willingness to participate in SCT clinical trials after receiving information [108]. Further, they observed that male gender was significantly correlated with positive SCT attitude (OR: 3.74, 95% CI: 1.45-9.61) [108]. Unsworth, et al., additionally identified younger age, greater perceived caregiver burden and poorer physical functioning, among others, as strong predictors for considering experimental SCT [110]. It is therefore clear that clinicians must place due emphasis on educating their SCT candidate populations to appreciate patient perspectives and ensure better-informed consent protocols in the high-risk neurosurgical arena.

Conclusion
Stroke remains a debilitating and costly sector of the healthcare system with limited options for intervention and rehabilitation. Due to their unique ability of selective differentiation, stem cells provide a great opportunity to bridge the gap in treatment for these patients. Invitro and in-vivo studies have already demonstrated promise in the use of MSC, BMSC and NSC with greater studies on the horizon. Through the use of exosomes and nanoparticles, intravenous and intrathecal delivery of stem cells may soon provide therapeutic benefit to those suffering from cognitive and neurological impairments following a stroke.